US7651999B2 - 2′, 5′-oligoadenylate analogs - Google Patents

2′, 5′-oligoadenylate analogs Download PDF

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US7651999B2
US7651999B2 US11/131,412 US13141205A US7651999B2 US 7651999 B2 US7651999 B2 US 7651999B2 US 13141205 A US13141205 A US 13141205A US 7651999 B2 US7651999 B2 US 7651999B2
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Makoto Koizumi
Koji Morita
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Sankyo Co Ltd
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/31—Chemical structure of the backbone
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    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
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    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to analogs of 2′,5′-oligoadenylate (2-5A) that are stable and have superior activity (particularly antitumor activity).
  • 2-5A which is known as a biological substance that has antiviral activity (Pharmacol. Ther. Vol. 78, No. 2, pp. 55-113, 1998), is a short-chain oligonucleotide composed of three or more adenosine units in which two adenosine 2′ and 5′ hydroxyl groups are linked with phosphate 2′,5′-phosphodiester bonds, and in which a triphosphate group is bonded to the 5′ end.
  • 2-5A synthetase is induced in the presence of viral dsRNA, and 2-5A is produced from ATP.
  • 2-5A is a substance that converts the inactive form of the RNA degrading enzyme, RNase L, into the active form within host cells. This activated RNase L inhibits viral growth in cells by degrading viral RNA. Moreover, when ovarian cancer cells Hey1B are transfected with 2-5A, sequence-specific cleavage of 18S rRNA is known to occur, that results in demonstration of antitumor activity as a result of apoptosis through release of cytochrome c and activation of caspase (J. Interferon Cytokine Res., 20, 1091-1100 (2000)). Thus, 2-5A is expected to act as a virus growth inhibitor, and, more specifically, as an antivirus drug or antitumor drug.
  • RNase L RNA degrading enzyme
  • an oligonucleotide composed of three or more adenosine units having a monophosphate group on the 5′ end and linked with 2′-5′ phosphodiester bonds is known to activate RNase L (Pharmacol. Ther. Vol. 78, No. 2, pp. 55-113, 1998; J. Biol. Chem. Vol. 270, No. 11, pp. 5963-5978 (1995)).
  • 2-5A itself is easily degraded to AMP and ATP by 2′-phosphodiesterase and nuclease.
  • the 5′-phosphate group or 5′-triphosphate group ends up being dephosphorylated by phosphatases in the living body and losing activity.
  • a 2-5A analog is desirable that has similar activity, but has high stability, making it more resistant to degradation and metabolism in the living body.
  • Y 1 and Y 2 represent a hydrogen atom or a protecting group for a hydroxy group
  • A represents an alkylene group having from 1 to 3 carbon atoms.
  • a 2-5A molecule bonded by means of a linker with an antisense molecule in the form of an oligonucleotide having a sequence complementary to mRNA involved in diseases has been used as a 2-5A antisense oligonucleotide that inhibits the function of mRNA (S. A. Adah, et al., Current Medicinal Chemistry (2001), 8, 1189-1212).
  • a highly stable 2-5A analog that is resistant to degradation and metabolism in the living body serves as a portion of a superior 2-5A antisense oligonucleotide, and is expected to be a useful drug.
  • oligonucleotides containing a bridged nucleoside in which an oxygen atom at the 2′ position and a carbon atom at the 4′ position of the sugar portion are bonded with an alkylene group are known to be useful as antisense molecules (Japanese Patent Application (Kokai) No. Hei 10-304889, Japanese Patent Application (Kokai) No. 2000-297097).
  • the inventors of the present invention conducted extensive research over the course of many years on non-natural type 2-5A analogs that have antivirus activity, antitumor activity or superior antisense activity, are stable in the living body, and are associated with the occurrence of few adverse side effects. As a result, they were found to be useful as stable and superior antivirus drugs, antitumor drugs and antisense drugs, thereby leading to completion of the present invention.
  • the 2-5A analog of the present invention relates to a 2′,5′-oligoadenylate analog represented by the general formula (1):
  • B represents a purin-9-yl group or a substituted purin-9-yl group having substituent(s) selected from the following Group ⁇
  • A represents an alkylene group having from 1 to 4 carbon atoms
  • D represents an alkyl group having from 1 to 6 carbon atoms which may be substituted, or an alkenyl group having from 2 to 6 carbon atoms which may be substituted
  • X 1 represents an alkyl group having from 1 to 24 carbon atoms which may be substituted, or an aryl group which may be substituted, or an aralkyl group which may be substituted
  • X 2 represents a —C( ⁇ O)O—, OC( ⁇ O)—, —C( ⁇ O)NH—, —NHC( ⁇ O)—, —C( ⁇ O)S—, —SC( ⁇ O)—, —OC( ⁇ O)NH—, —NHC( ⁇ O)O—, —NHC( ⁇ O)NH—, —OC( ⁇ S)
  • the above 2′,5′-oligoadenylate analog or pharmacologically acceptable salt thereof is preferably
  • R 1 is an alkoxy group having from 1 to 4 carbon atoms which may be substituted, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, or an alkylthio group having from 1 to 4 carbon atoms which may be substituted, or a group of formula: X 1 —X 2 —X 3 —S—;
  • R 2 , R 3 , R 4 , R 5 and R 6 represent a hydroxyl group, a hydroxyl group protected by a nucleic acid synthesis protecting group, an alkoxy group having from 1 to 4 carbon atoms which may be substituted, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, an alkylthio group having from 1 to 4 carbon atoms which may be substituted, or a group of formula: X 1 —X 2 —
  • FIG. 1 is a graph showing the cytotoxic activity on A549 cells as a result of the addition of compounds, namely, natural type 2-5A, the compound of Example 1 (Exemplary Compound No. 4), the compound of Example 2 (Exemplary Compound No. 1), the compound of Example 3 (Exemplary Compound No. 5) and the compound of Example 4 (Exemplary Compound No. 8).
  • the “alkylene group having from 1 to 4 carbon atoms” of A can be, for example, a methylene, ethylene, trimethylene or tetramethylene group, and is preferably an ethylene or trimethylene group.
  • the protecting group of the “hydroxyl group protected by a nucleic acid synthesis protecting group” of R 2 , R 3 , R 4 , R 5 and R 6 or the Group ⁇ is not particularly limited so long as it can stably protect a hydroxyl group during nucleic acid synthesis, and specifically means a protecting group stable under acidic or neutral conditions, and cleavable by a chemical method such as hydrogenolysis, hydrolysis, electrolysis or photolysis.
  • Such a protecting group can be, for example, an “aliphatic acyl group” such as an alkylcarbonyl group, e.g., formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexadecano
  • a “lower alkyl group” such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl and 2-ethylbutyl; a “lower alkenyl group” such as ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-
  • the protecting group of the “hydroxyl group protected by a nucleic acid synthesis protecting group” of R 2 , R 3 , R 4 , R 5 and R 6 or the Group ⁇ is preferably a “methyl group substituted by from 1 to 3 aryl groups”, an “aryl group substituted by halogen atom(s), lower alkoxy group(s) or nitro group(s)”, a “lower alkyl group”, a “lower alkenyl group”, an “aliphatic acyloxymethyl group”, or an “aliphatic acylthioethyl group”, more preferably a benzyl group, a 2-chlorophenyl group, a 4-chlorophenyl group, a 2-propenyl group, a pivaloyloxymethyl group, an acetylthioethyl group, or a pivaloylthioethyl group.
  • the “alkoxy group having from 1 to 6 carbon atoms which may be substituted” of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 or the Group ⁇ can be, for example, a “lower alkyloxy group” such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, n-pentyloxy, isopentyloxy, 2-methylbutoxy, neopentyloxy, 1-ethylpropoxy, n-hexyloxy, isohexyloxy, 4-methylpentyloxy, 3-methylpentyloxy, 2-methylpentyloxy, 1-methylpentyloxy, 3,3-dimethylbutoxy, 2,2-dimethylbutoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,3-dimethylbutoxy and 2-ethy
  • a “lower alkyloxy group substituted by hydroxyl group(s)” such as 1-hydroxymethyloxy, 2-hydroxyethyloxy, 3-hydroxypropyloxy, 4-hydroxybutyloxy, 2-hydroxypropyloxy, 1-methyl-2-hydroxyethyloxy, 1-methyl-1-hydroxyethyloxy, 1,1-dimethyl-2-hydroxyethyloxy, 2-hydroxybutyloxy, 3-hydroxybutyloxy, 1-methyl-3-hydroxypropyloxy and 2-methyl-3-hydroxypropyloxy;
  • a “lower alkyloxy group substituted by amino group(s)” such as 1-aminomethyloxy, 2-aminoethyloxy, 3-aminopropyloxy, 4-aminobutyloxy, 2-aminopropyloxy, 1-methyl-2-aminoethyloxy, 1-methyl-1-aminoethyloxy, 1,1-dimethyl-1-aminoethyloxy, 2-aminobutyloxy, 3-aminobutyl
  • the “oxyalkyleneoxy group having from 1 to 6 carbon atoms” of R 7 can be, for example, an oxymethyleneoxy, oxyethyleneoxy, oxytrimethyleneoxy, oxytetramethyleneoxy, oxypentamethyleneoxy, or oxyhexamethyleneoxy group, and is preferably an oxytetramethyleneoxy or oxypentamethyleneoxy group.
  • the protecting group of the “mercapto group protected by a nucleic acid synthesis protecting group” of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 or the Group ⁇ is not particularly limited so long as it can stably protect a mercapto group during nucleic acid synthesis, and specifically means a protecting group stable under acidic or neutral conditions, and cleavable by a chemical method such as hydrogenolysis, hydrolysis, electrolysis or photolysis.
  • Such a protecting group can be, for example, a “group which can form a disulfide” such as an alkylthio group, e.g., methylthio, ethylthio and tert-butylthio, or an arylthio group, e.g.
  • benzylthio in addition to the groups listed as a protecting group of a hydroxyl group, and is preferably an “aliphatic acyl group”, an “aromatic acyl group”, an “aliphatic acyloxymethyl group”, or an “aliphatic acylthioethyl group”, more preferably a pivaloyloxymethyl group, an acetylthioethyl group, or a pivaloylthioethyl group.
  • the “alkylthio group having from 1 to 4 carbon atoms which may be substituted” of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 or the Group ⁇ can be, for example, methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, s-butylthio, or tert-butylthio, and is preferably a methylthio or ethylthio group.
  • the protecting group of the “amino group protected by a nucleic acid synthesis protecting group” of R 1 R 2 , R 3 , R 4 , R 5 and R 6 or the Group ⁇ is not particularly limited so long as it can stably protect an amino group during nucleic acid synthesis, and specifically means a protecting group stable under acidic or neutral conditions and cleavable by a chemical method such as hydrogenolysis, hydrolysis, electrolysis or photolysis.
  • Such a protecting group can be, for example, an “aliphatic acyl group” such as an alkylcarbonyl group, e.g., formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexadecano
  • an “aromatic acyl group” such as an arylcarbonyl group, e.g., benzoyl, ⁇ -naphthoyl and ⁇ -naphthoyl; a halogeno arylcarbonyl group, e.g., 2-bromobenzoyl and 4-chlorobenzoyl; a lower alkylated arylcarbonyl group, e.g., 2,4,6-trimethylbenzoyl and 4-toluoyl; a lower alkoxylated arylcarbonyl group, e.g., 4-anisoyl; a carboxylated arylcarbonyl group, e.g., 2-carboxybenzoyl, 3-carboxybenzoyl and 4-carboxybenzoyl; a nitrated arylcarbonyl group, e.g., 4-nitrobenzoyl and 2-nitrobenzoyl; a lower alkoxycarbonylated
  • lower alkoxycarbonyl group such as methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl and isobutoxycarbonyl
  • lower alkoxycarbonyl group substituted by halogen or tri-lower alkylsilyl group(s) such as 2,2,2-trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl;
  • alkenyloxycarbonyl group such as vinyloxycarbonyl and allyloxycarbonyl
  • an “aralkyloxycarbonyl group whose aryl ring may be substituted by 1 or 2 lower alkoxy or nitro groups” such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl and 4-nitrobenzyloxycarbonyl; and is preferably an “aliphatic acyl group” or an “aromatic acyl group”, more preferably a benzoyl group.
  • the “amino group substituted by alkyl group(s) having from 1 to 4 carbon atoms which may be substituted” of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 or the Group ⁇ can be, for example, a “lower alkylamino group” such as methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, s-butylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di(s-butyl)amino and di(tert-butyl)amino;
  • a “lower alkylamino group” such as methylamino, ethylamino, propylamino, isopropylamino, but
  • a “lower alkylamino group substituted by hydroxyl group(s), lower alkoxy group(s) or halogen atom(s)” such as 1-hydroxyethylamino, 2-hydroxyethylamino, 1-methoxyethylamino, 2-methoxyethylamino, 1-bromoethylamino, 2-methoxyethylamino, 1-chloroethylamino and 2-chloroethylamino; or a “lower alkoxycarbonylamino group” such as 1-methoxycarbonylethylamino, 2-methoxycarbonylethylamino, 1-ethoxycarbonylethylamino, 2-ethoxycarbonylethylamino, 1-propoxycarbonylethylamino and 1-propoxycarbonylethylamino; and is preferably a 1-hydroxyethylamino, 2-hydroxyethylamino, methylamino, ethyla
  • the “alkyl group having from 1 to 6 carbon atoms which may be substituted” of D, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 8 or the Group ⁇ can be, for example, a “lower alkyl group” such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbut
  • a “lower alkyl group substituted by hydroxyl group(s)” such as 1-hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, 2-hydroxypropyl, 1-methyl-2-hydroxyethyl, 1-methyl-1-hydroxyethyl, 1,1-dimethyl-2-hydroxyethyl, 2-hydroxybutyl, 3-hydroxybutyl, 1-methyl-3-hydroxypropyl and 2-methyl-3-hydroxypropyl;
  • a “lower alkyl group substituted by amino group(s)” such as 1-aminomethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 2-aminopropyl, 1-methyl-2-aminoethyl, 1-methyl-1-aminoethyl, 1,1-dimethyl-2-aminoethyl, 2-aminobutyl, 3-aminobutyl, 1-methyl-3-aminopropyl and 2-methyl-3-aminopropyl;
  • the “alkyl group having from 1 to 24 carbon atoms which may be substituted” of X 1 can be, for example, stearyl, 2,2-dimethylstearyl, heptadecyl, 2,2-dimethylheptadecyl, hexadecyl, 2,2-dimethylhexadecyl, pentadecyl, 2,2-dimethylpentadecyl, tetradecyl, 2,2-dimethyltetradecyl, tridecyl, 2,2-dimethyltridecyl, dodecyl, 2,2-dimethyldodecyl, undecyl, 2,2-dimethylundecyl, decyl, 2,2-dimethyldecyl, nonyl, 2,2-dimethylnonyl, octyl, 2,2-dimethyloctyl, heptyl, 2,2-dimethylheptyl, hexy
  • the “alkylene group having from 1 to 6 carbon atoms which may be substituted” of X 3 can be, for example, methylene, ethylene, propylene, butylene, 2,2-dimethylethylene, 2,2-dimethylpropylene, or 2,2-dimethylbutylene, and is preferably methylene or ethylene.
  • the “aryloxy group which may be substituted” of R 1 can be, for example, an “aryloxy group substituted by lower alkyl group(s), halogen atom(s) or nitro group(s)” such as 2-methylphenoxy, 3-methylphenoxy, 4-methylphenoxy, 2,6-dimethylphenoxy, 2-chlorophenoxy, 4-chlorophenoxy, 2,4-dichlorophenoxy, 2,5-dichlorophenoxy, 2-bromophenoxy, 4-nitrophenoxy and 4-chloro-2-nitrophenoxy.
  • an “aryloxy group substituted by lower alkyl group(s), halogen atom(s) or nitro group(s)” such as 2-methylphenoxy, 3-methylphenoxy, 4-methylphenoxy, 2,6-dimethylphenoxy, 2-chlorophenoxy, 4-chlorophenoxy, 2,4-dichlorophenoxy, 2,5-dichlorophenoxy, 2-bromophenoxy, 4-nitrophenoxy and 4-chloro-2-nitrophenoxy.
  • the “aryl group which may be substituted” of R 8 or X 1 can be, for example, an “aryl group substituted by lower alkyl group(s), halogen atom(s) or nitro group(s)” such as 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, 2-bromophenyl, 4-nitrophenyl and 4-chloro-2-nitrophenyl.
  • an “aryl group substituted by lower alkyl group(s), halogen atom(s) or nitro group(s)” such as 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, 2-brom
  • the “arylthio group which may be substituted” of R 1 can be, for example, an “arylthio group substituted by lower alkyl group(s), halogen atom(s) or nitro group(s)” such as 2-methylphenylthio, 3-methylphenylthio, 4-methylphenylthio, 2,6-dimethylphenylthio, 2-chlorophenylthio, 4-chlorophenylthio, 2,4-dichlorophenylthio, 2,5-dichlorophenylthio, 2-bromophenylthio, 4-nitrophenylthio and 4-chloro-2-nitrophenylthio.
  • an “arylthio group substituted by lower alkyl group(s), halogen atom(s) or nitro group(s)” such as 2-methylphenylthio, 3-methylphenylthio, 4-methylphenylthio, 2,6-dimethylphenylthio, 2-chlorophenylthio
  • the “alkenyl group having from 2 to 6 carbon atoms which may be substituted” of D can be, for example, ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 2-ethyl-2-propenyl, 1-butenyl, 2-butenyl, 1-methyl-2-butenyl, 1-methyl-1-butenyl, 3-methyl-2-butenyl, 1-ethyl-2-butenyl, 3-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 1-ethyl-3-butenyl, 1-pentenyl, 2-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 4-pentenyl, 1-methyl-4-
  • the “aralkyl group which may be substituted” of R 8 or X 1 can be, for example, an “aralkyl group” such as benzyl, ⁇ -naphthylmethyl, ⁇ -naphthylmethyl, indenylmethyl, phenanthrenylmethyl, anthracenylmethyl, diphenylmethyl, triphenylmethyl, 1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl, 3-naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl, 4-phenylbutyl, 1-naphthylbutyl, 2-naphthylbutyl, 2-
  • the preferred groups are 6-amino-purin-9-yl (that is, adeninyl), 6-amino-purin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 6-amino-8-bromopurin-9-yl, 6-amino-8-bromopurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 6-amino-8-chloropurin-9-yl, 6-amino-8-chloropurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 6-amino-8-fluoropurin-9-yl, 6-amino-8-fluoropurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 6-amino-8-methoxypur
  • X 1 —X 2 —X 3 —S there is no particular limitation on the functional group represented by “X 1 —X 2 —X 3 —S”, provided that it is a combination comprising X 1 , X 2 , X 3 and S mentioned above, and it can be, for example, an acyloxyalkylthio group such as 2-(stearoyloxy)ethylthio, 2-(myristoyloxy)ethylthio, 2-(decanoyloxy)ethylthio, 2-(benzoyloxy)ethylthio, 2-(pivaloyloxy)ethylthio, 2-(2,2-dimethyloctadecanoyloxy)ethylthio, 3-(stearoyloxy)propylthio, 3-(myristoyloxy)propylthio, 3-(decanoyloxy)propylthio, 3-(benzoyloxy)propylthio, 3-(pivalo
  • the “halogen atom” of the Group ⁇ can be, for example, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and is preferably a bromine atom or a chlorine atom.
  • the “2′,5′-oligoadenylate analog (2-5A analog)” means a non-natural type derivative of “2′,5′-oligoadenylate”, in which the 2′ position and 5′ position of the 3 or 4 “nucleosides”, being the same or different, are bonded by a phosphodiester bond linkage or a modified phosphodiester linkage, and a phosphoryl derivative is bonded to the 5′-terminal, or a phosphoryl derivative is optionally bonded to the 2′-terminal, or a 5′-phosphorylated oligonucleotide analog is optionally bonded to the 2′-terminal through an alkylene linker.
  • Such an analog can preferably be a sugar derivative wherein the sugar portion is modified; a thioate derivative wherein the phosphodiester bonding portion is thioated; a phosphoryl derivative wherein the phosphoric acid portion at the terminal is substituted; or a purine derivative wherein the purine base is substituted; and is more preferably a phosphoryl derivative wherein the phosphoric acid portion at the terminal is substituted, a sugar derivative wherein the sugar portion is modified, or a thioate derivative wherein the phosphodiester bonding portion is thioated.
  • the “5′-phosphorylated oligonucleotide analog which has one hydroxyl group removed from the 5′-phosphoric acid group” means a non-natural type derivative of “oligonucleotide” in which 2 to 50 “nucleosides” being the same or different, are bonded by phosphodiester bond linkages, and means a derivative having the following residual group:
  • Such an analog can preferably be a sugar derivative wherein the sugar portion is modified; a thioate derivative wherein the phosphodiester bonding portion is thioated; an ester wherein the phosphoric acid portion at the terminal is esterified; or an amide wherein the amino group on the purine base is amidated; and is more preferably a sugar derivative wherein the sugar portion is modified, or a thioate derivative wherein the phosphodiester bonding portion is thioated.
  • Salt thereof means a salt of the compound (1) of the present invention, since the compound can be converted to a salt.
  • a salt can preferably be a metal salt such as an alkali metal salt, e.g., a sodium salt, a potassium salt and a lithium salt; an alkaline earth metal salt, e.g., a calcium salt and a magnesium salt; an aluminum salt, an iron salt, a zinc salt, a copper salt, a nickel salt or a cobalt salt; an amine salt such as inorganic salt, e.g., an ammonium salt; or an organic salt, e.g., a t-octylamine salt, a dibenzylamine salt, a morpholine salt, a glucosamine salt, a phenylglycine alkyl ester salt, an ethylenediamine salt, an N-methylglucamine salt, a guanidine salt, a diethylamine salt, a triethyl
  • a “pharmacologically acceptable salt thereof” means a salt of the 2-5A analog of the present invention, since it can be converted into a salt.
  • a salt can preferably be a metal salt such as an alkali metal salt, e.g., a sodium salt, a potassium salt and a lithium salt; an alkaline earth metal salt, e.g., a calcium salt and a magnesium salt; an aluminum salt, an iron salt, a zinc salt, a copper salt, a nickel salt or a cobalt salt; an amine salt such as an inorganic salt, e.g., an ammonium salt; or an organic salt; e.g., a t-octylamine salt, a dibenzylamine salt, a morpholine salt, a glucosamine salt, a phenylglycine alkyl ester salt, an ethylenediamine salt, an N-methylglucamine salt, a guanidine salt, a diethylamine
  • Ph represents a phenyl group
  • Bn represents a benzyl group
  • Me represents a methyl group
  • Et represents an ethyl group
  • Pr represents an n-propyl group
  • tBu represents a tert-butyl group
  • the groups described as K x represent groups having the following structure.
  • the groups described as Gly, POMO, POMS, ATE, PTE, ALM, L 1 , L 2 , C 20 , C 18 , C 14 and C 10 represent groups having the following structures respectively.
  • the groups described as ON X represent oligonucleotide analogs having the structures defined below and bonded to R 7 at the terminal.
  • groups described as A n , G n , C n , T n , A e , G e , C e , T e , p, s and hp in the above represent groups having the following structure.
  • ON 1 is a sequence in human telomerase (GenBank Accession No. U86046, base sequence of the complementary chain of nucleotide numbers 170 to 188)
  • ON 2 is a sequence in human breakpoint cluster region (BCR) mRNA (GenBank Accession No. NM-021574.1, base sequence of the complementary chain of nucleotide numbers 597 to 614)
  • ON 3 is a sequence in interferon-inducible double-stranded RNA-dependent human protein kinase (PKR) mRNA (GenBank Accession No.
  • ON 4 is a sequence in human protein kinase C, alpha (PKC ⁇ ) mRNA (GenBank Accession No. NM-002737.1, base sequence of the complementary chain of nucleotide numbers 2044 to 2063)
  • ON 5 is a sequence in human intercellular adhesion molecule (ICAM1) mRNA (GenBank Accession No. NM-000201.1, base sequence of the complementary chain of nucleotide numbers 2100 to 2119)
  • ON 6 is a sequence in human ras transforming protein gene (GenBank Accession No.
  • ON 7 is a sequence in human tumor necrosis factor (TNF superfamily, member 2) (TNF) mRNA (GenBank Accession No. NM-000594.1, base sequence of the complementary chain of nucleotide numbers 279 to 298)
  • ON 8 is a sequence in human phosphotyrosyl-protein phosphatase (PTP-1B) mRNA (GenBank Accession No. M31724.1, base sequence of the complementary chain of nucleotide numbers 951 to 970)
  • ON 9 is a sequence in human c-raf-1 mRNA (GenBank Accession No.
  • NM-002880.1 base sequence of the complementary chain of nucleotide numbers 2484 to 2503), and ON 10 is a sequence in human telomerase mRNA (GenBank Accession No. U86046, base sequence of the complementary chain of nucleotide numbers 136 to 148).
  • the preferred compounds are 1, 2, 3, 4, 5, 6, 7, 8, 13, 22, 27, 28, 31, 39, 41, 42, 50, 52, 53, 61, 63, 64, 71, 73, 77, 79, 96, 98, 102, 104, 146, 148, 152, 154, 171, 173, 177, 179, 290, 292, 293, 305, 307, 310, 311, 312, 313, 314, 316, 319, 320, 325, 330, 334, 338, 339, 343, 344, 351, 356, 364, 369, 377, 382, 386, 390, 391, 395, 396, 403, 408, 416, 421, 424, 425, 428, 438, 441, 451, 452, 453, 454, 455, 461, 462, 463, 464, 465, 471, 472, 473, 474, 475, 481, 482, 483, 484, 485, 491, 492, 493, 494, 495, 501, 50
  • the compounds (1) of the present invention can be prepared by appropriately utilizing Process A, Process B, Process C, Process D, Process E, Process F, Process G, and Process H mentioned below.
  • A, D, R 1 , R 7 , and R 8 have the same meanings as defined above;
  • R 9 represents a protecting group for protecting a phosphoric acid group or a phosphorous acid group;
  • R 10 represents a dialkylamino group (particularly a diisopropylamino group or a diethylamino group);
  • R 11 represents an R 1 group which requires a protecting group in the synthesis of the 2-5A analog;
  • B 1 represents a purin-9-yl group or a substituted purin-9-yl group having substituent(s) selected from the above Group ⁇ , but a group substituted by amino group is excluded.
  • R 12 and R 16 are the same or different and represent a protecting group
  • R 13 represents a —(CH 2 )h- group (h is an integer of from 2 to 8)
  • R 14 represents a hydroxyl group, a phenyloxy group which may be substituted, or an ethyloxy group which may be substituted by halogen
  • R 15 represents an oxygen atom, a sulfur atom or an NH group
  • HR 15 —P (encircled) represents a high molecular weight compound.
  • the “protecting group” in the definition of R 9 can be, for example, a lower alkyl group such as methyl; a lower alkenyl group such as 2-propenyl; a cyano lower alkyl group such as 2-cyanoethyl; a lower alkoxylated lower alkoxymethyl group such as 2-methoxyethoxymethyl; a halogeno lower alkoxymethyl group such as 2,2,2-trichloroethoxymethyl and bis(2-chloroethoxy)methyl; a halogenated ethyl group such as 2,2,2-trichloroethyl; a methyl group substituted by an aryl group such as benzyl; a methyl group substituted by from 1 to 3 aryl groups whose aryl ring is substituted by lower alkyl, lower alkoxy, halogen or cyano group(s) such as 4-methylbenzyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-brom
  • the “protecting group” in the definition of R 12 and R 16 can be, for example, an “acyl type” protecting group including an “aliphatic acyl group” such as an alkylcarbonyl group, e.g., formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl,
  • an “aromatic acyl group” such as an arylcarbonyl group, e.g., benzoyl, ⁇ -naphthoyl and ⁇ -naphthoyl; a halogeno arylcarbonyl group, e.g., 2-bromobenzoyl and 4-chlorobenzoyl; a lower alkylated arylcarbonyl group, e.g., 2,4,6-trimethylbenzoyl and 4-toluoyl; a lower alkoxylated arylcarbonyl group, e.g., 4-anisoyl; a carboxylated arylcarbonyl group, e.g., 2-carboxybenzoyl, 3-carboxybenzoyl and 4-carboxybenzoyl; a nitrated arylcarbonyl group, e.g., 4-nitrobenzoyl and 2-nitrobenzoyl; a lower alkoxycarbonylated
  • a “lower alkyl group” such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl and 2-ethylbutyl; a “lower alkenyl group” such as ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-
  • the present step is a step, wherein compound (3) is produced by reacting compound (2) with a mono-substituted chloro(alkoxy)phosphine, di-substituted alkoxyphosphine, mono-substituted chloro(benzyloxy)phosphine, or di-substituted benzyloxyphosphine normally used for amidite formation, in an inert solvent.
  • the solvent to be used is not particularly limited so long as it does not affect the reaction, but can preferably be an ether such as tetrahydrofuran, diethyl ether or dioxane; or a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene or dichlorobenzene.
  • an ether such as tetrahydrofuran, diethyl ether or dioxane
  • a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene or dichlorobenzene.
  • the mono-substituted chloro(alkoxy)phosphine to be used can be, for example, a phosphine such as chloro(morpholino)methoxyphosphine, chloro(morpholino)cyanoethoxyphosphine, chloro(dimethylamino)methoxyphosphine, chloro(dimethylamino)cyanoethoxyphosphine, chloro(diisopropylamino)methoxyphosphine or chloro(diisopropylamino)cyanoethoxyphosphine, and is preferably chloro(morpholino)methoxyphosphine, chloro(morpholino)cyanoethoxyphosphine, chloro(diisopropylamino)methoxyphosphine or chloro(diisopropylamino)cyanoethoxyphosphine.
  • a phosphine such as chloro(morpholino)methoxyphos
  • a deoxidizer is used.
  • the deoxidizer to be used can be a heterocyclic amine such as pyridine or dimethylaminopyridine; or an aliphatic amine such as trimethylamine, triethylamine, diisopropylamine or diisopropylethylamine, and is preferably an aliphatic amine (particularly diisopropylethylamine).
  • the di-substituted alkoxyphosphine to be used can be, for example, a phosphine such as bis(diisopropylamino)cyanoethoxyphosphine, bis(diethylamino)methanesulfonylethoxyphosphine, bis(diisopropylamino)(2,2,2-trichloroethoxy)phosphine or bis(diisopropylamino)(4-chlorophenylmethoxy)phosphine, and is preferably bis(diisopropylamino)cyanoethoxyphosphine.
  • a phosphine such as bis(diisopropylamino)cyanoethoxyphosphine, bis(diethylamino)methanesulfonylethoxyphosphine, bis(diisopropylamino)(2,2,2-trichloroethoxy)phosphine or bis(
  • an acid or an organic salt is used.
  • the acid to be used is tetrazol, acetic acid or p-toluenesulfonic acid
  • the organic salt to be used is tetrazol diisopropylamine salt, acetic acid diisopropylamine salt or p-toluenesulfonic acid diisopropylamine salt, preferably tetrazol or tetrazol diisopropylamine salt.
  • the mono-substituted chloro(benzyloxy)phosphine to be used can be, for example, a phosphine such as chloro(morpholino)benzyloxyphosphine, chloro(dimethylamino)methoxyphosphine, chloro(dimethylamino)benzyloxyphosphine or chloro(diisopropylamino)benzyloxyphosphine, and is preferably chloro(diisopropylamino)benzyloxyphosphine.
  • a phosphine such as chloro(morpholino)benzyloxyphosphine, chloro(dimethylamino)methoxyphosphine, chloro(dimethylamino)benzyloxyphosphine or chloro(diisopropylamino)benzyloxyphosphine, and is preferably chloro(diisopropylamino)benzyloxyphosphine.
  • a deoxidizer is used.
  • the deoxidizer to be used can be a heterocyclic amine such as pyridine or dimethylaminopyridine; or an aliphatic amine such as trimethylamine, triethylamine, diisopropylamine or diisopropylethylamine, and is preferably an aliphatic amine (particularly diisopropylethylamine).
  • the di-substituted benzyloxyphosphine to be used can be, for example, a phosphine such as bis(diisopropylamino)benzyloxyphosphine or bis(diethylamino)benzyloxyphosphine, and is preferably bis(diisopropylamino)benzyloxyphosphine.
  • a phosphine such as bis(diisopropylamino)benzyloxyphosphine or bis(diethylamino)benzyloxyphosphine, and is preferably bis(diisopropylamino)benzyloxyphosphine.
  • an acid or an organic salt is used.
  • the acid to be used is tetrazol, acetic acid or p-toluenesulfonic acid
  • the organic salt to be used is tetrazol diisopropylamine salt, acetic acid diisopropylamine salt or p-toluenesulfonic acid diisopropylamine salt, preferably tetrazol or tetrazol diisopropylamine salt.
  • the reaction temperature is not particularly limited, but is normally from 0 to 80° C., preferably room temperature.
  • reaction time varies depending on the starting materials, the reagents and the temperature used, it is normally from 5 minutes to 30 hours; and in the case where the reaction is carried out at room temperature, it is preferably from 30 minutes to 10 hours.
  • the desired compound (3) of the present reaction is obtained, for example, by, after suitably neutralizing the reaction mixture, and removing any insoluble matter, if present, by filtration, addition of water and an immiscible organic solvent such as ethyl acetate, followed by washing with water, separating the organic layer containing the desired compound, drying with anhydrous magnesium sulfate or the like, and distilling off the solvent.
  • the thus obtained desired compound can be further purified by ordinary methods such as recrystallization, reprecipitation or chromatography, if necessary.
  • the present step is a step, wherein compound (4) is produced by allowing compound (2) to react with tris-(1,2,4-triazolyl)phosphite in an inert solvent (preferably a halogenated hydrocarbon such as methylene chloride), and adding water thereto to cause H-phosphonation.
  • an inert solvent preferably a halogenated hydrocarbon such as methylene chloride
  • the reaction temperature is not particularly limited, but is normally from ⁇ 20 to 100° C., preferably from 10 to 40° C.
  • reaction time varies depending on the starting materials, the reagents and the temperature used, it is normally from 5 minutes to 30 hours; and in the case where the reaction is carried out at room temperature, it is preferably 30 minutes.
  • the desired compound (4) of the present reaction is obtained, for example, by, after suitably neutralizing the reaction mixture, and removing any insoluble matter, if present, by filtration, addition of water and an immiscible organic solvent such as ethyl acetate, followed by washing with water, separating the organic layer containing the desired compound, drying with anhydrous magnesium sulfate or the like, and distilling off the solvent.
  • the thus obtained desired compound can be further purified by ordinary methods such as recrystallization, reprecipitation or chromatography, if necessary.
  • the present step is a step, wherein compound (5) is produced by allowing compound (2) to react with a bis(1,2,4-triazolyl)arylphosphate, bis(1,2,4-triazolyl)benzylphosphate, bis(1,2,4-triazolyl)-2-cyanoethylphosphate, bis(1,2,4-triazolyl)(2,2,2-trichloroethyl)phosphate or bis(1,2,4-triazolyl)(2-propenyl)phosphate in an inert solvent (preferably a halogenated hydrocarbon such as methylene chloride), and adding water thereto to make a phosphodiester.
  • an inert solvent preferably a halogenated hydrocarbon such as methylene chloride
  • the bis(1,2,4-triazolyl)arylphosphate to be used can be, for example, bis(1,2,4-triazolyl)phenylphosphate, bis(1,2,4-triazolyl)(2-chlorophenyl)phosphate, bis(1,2,4-triazolyl)(4-chlorophenyl)phosphate, bis(1,2,4-triazolyl)(2-nitrophenyl)phosphate or bis(1,2,4-triazolyl)(4-nitrophenyl)phosphate, and is preferably bis(1,2,4-triazolyl)(2-chlorophenyl)phosphate or bis(1,2,4-triazolyl)(4-chlorophenyl)phosphate.
  • the reaction temperature is not particularly limited, but is normally from ⁇ 20 to 100° C., preferably from 10 to 40° C.
  • reaction time varies depending on the starting materials, the reagents and the temperature used, it is normally from 5 minutes to 30 hours; and in the case where the reaction is carried out at room temperature, it is preferably 30 minutes.
  • the desired compound (5) of the present reaction is obtained, for example, by, after suitably neutralizing the reaction mixture, and removing any insoluble matter, if present, by filtration, addition of water and an immiscible organic solvent such as ethyl acetate, followed by washing with water, separating the organic layer containing the desired compound, drying with anhydrous magnesium sulfate or the like, and distilling off the solvent.
  • the thus obtained desired compound can be further purified by ordinary methods such as recrystallization, reprecipitation or chromatography, if necessary.
  • the present step is a step, wherein compound (7) is produced by allowing compound (6) to react with a protecting reagent in the presence of a basic catalyst in an inert solvent.
  • the solvent to be used can preferably be an aromatic hydrocarbon such as benzene, toluene or xylene; a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene or dichlorobenzene; an ester such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate or diethyl carbonate; an ether such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane or diethylene glycol dimethyl ether; a ketone such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone or cyclohexanone; a nitro compound such as nitroethane or nitrobenzene; a nitrile such as acetonit
  • the protecting reagent to be used is not particularly limited so long as it is adapted for the following nucleic acid synthesis and can be removed under acidic or neutral conditions, and can preferably be a tri-arylmethyl halide such as trityl chloride, mono-methoxytrityl chloride or dimethoxytrityl chloride; or a triarylmethanol ether such as dimethoxytrityl-O-triflate.
  • a base is normally used.
  • the base to be used can be a heterocyclic amine such as pyridine, dimethylaminopyridine or pyrrolidinopyridine; or an aliphatic tertiary amine such as trimethylamine or triethylamine; and is preferably pyridine, dimethylaminopyridine or pyrrolidinopyridine.
  • the reaction temperature varies depending on the starting materials, the reagents and the solvent used, and is normally from 0 to 150° C., preferably from 20 to 100° C. While the reaction time varies depending on the starting materials, the solvent and the reaction temperature used, it is normally from 1 to 100 hours, preferably from 2 to 24 hours.
  • the desired compound (7) of the present reaction is obtained, for example, by concentrating the reaction mixture, adding water and an immiscible organic solvent such as ethyl acetate, followed by washing with water, separating the organic layer containing the desired compound, drying with anhydrous magnesium sulfate or the like, and distilling off the solvent.
  • an immiscible organic solvent such as ethyl acetate
  • the resulting compound can be further purified by ordinary methods, for example, recrystallization or silica gel column chromatography, if necessary.
  • the present step is a step, wherein compound (8) is produced by allowing compound (7) prepared in Step B-1 to react with a mono-substituted chloro(alkoxy)phosphine, di-substituted alkoxyphosphine, mono-substituted chloro(benzyloxy)phosphine or di-substituted benzyloxyphosphine, which is normally used for amidite formation, in an inert solvent.
  • the present step is carried out similarly to Step (A-1).
  • the present step is a step, wherein compound (9) is produced by allowing compound (7) prepared in Step B-1 to react with tris-(1,2,4-triazolyl)phosphite in an inert solvent (preferably a halogenated hydrocarbon such as methylene chloride), followed by adding water to carry out H-phosphonation.
  • an inert solvent preferably a halogenated hydrocarbon such as methylene chloride
  • the present step is carried out similarly to Step (A-2).
  • the present step is a step, wherein compound (8) is produced by allowing compound (7) prepared in Step B-1 to react with a bis(1,2,4-triazolyl)arylphosphate, bis(1,2,4-triazolyl)benzylphosphate, bis(1,2,4-triazolyl)-2-cyanoethylphosphate, bis(1,2,4-triazolyl) (2,2,2-trichloroethyl)phosphate, or bis(1,2,4-triazolyl) (2-propenyl)phosphate in an inert solvent (preferably a halogenated hydrocarbon such as methylene chloride), followed by adding water to make a phosphodiester.
  • an inert solvent preferably a halogenated hydrocarbon such as methylene chloride
  • the present step is carried out similarly to Step A-3.
  • the present step is a step, wherein compound (12) is produced by allowing compound (11) to react with a mono-substituted chloro(alkoxy)phosphine, di-substituted alkoxyphosphine, mono-substituted chloro(benzyloxy)phosphine, or di-substituted benzyloxyphosphine normally used for amidite formation, in an inert solvent.
  • Compound (11) is a compound wherein a nucleoside has been reacted with an alkyl halide such as methyl iodide or an alkenyl halide such as allyl bromide in the presence of sodium hydride, according to the method described in PCT/US94/10131, to obtain the 3′-substituted compound, and then the 5′-hydroxyl group, and amino group of the base portion, have been protected by protecting groups.
  • an alkyl halide such as methyl iodide or an alkenyl halide such as allyl bromide
  • 3′-O-allyladenosine (catalogue No.: RP-3101) can be purchased from ChemGene Industries, and 5′-O-dimethoxytrityl-3′-O-allyl-N-benzoyladenosine can be obtained therefrom by protection using publicly known methods.
  • the present step is carried out similarly to Step A-1.
  • 5′-O-dimethoxytrityl-3′-O-methyl-N-benzoyladenosine-2′-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) (catalogue No.: ANP-2901), for example, can be purchased from ChemGene Industries.
  • the present step is a step, wherein compound (14) is produced by allowing compound (13) to react with a mono-substituted chloro(alkoxy)phosphine, di-substituted alkoxyphosphine, mono-substituted chloro(benzyloxy)phosphine, or di-substituted benzyloxyphosphine normally used for amidite formation, in an inert solvent.
  • Compound (13) is the same compound as compound (20) described in Process F of Japanese Patent Application (Kokai) No. 2002-249497, or the compound described in Japanese Patent Application (Kokai) No. Hei 10-195098 in which Y 1 is a protecting group and Y 2 is a hydrogen atom.
  • the present step is carried out similarly to Step (A-1).
  • the present step is a step, wherein compound (16) is produced by allowing compound (15) to react with a protecting reagent in the presence of a basic catalyst in an inert solvent.
  • the present step is carried out similarly to Step (B-1).
  • the present step is a step, wherein compound (17) is produced by allowing compound (16) prepared in Step E-1 to react with a dicarboxylic anhydride in an inert solvent.
  • the solvent to be used is not particularly limited so long as it does not inhibit the reaction and dissolves the starting material to a certain extent, and can be, for example, an aromatic hydrocarbon such as benzene, toluene or xylene; a halogenated hydrocarbon such as methylene chloride or chloroform; an ether such as ether, tetrahydrofuran, dioxane or dimethoxyethane; an amide such as dimethylformamide, dimethylacetamide or hexamethylphosphortriamide; a sulfoxide such as dimethyl sulfoxide; a ketone such as acetone or methyl ethyl ketone; a heterocyclic amine such as pyridine; or a nitrile such as acetonitrile; and is preferably a halogenated hydrocarbon such as methylene chloride.
  • an aromatic hydrocarbon such as benzene, toluene or xylene
  • the deoxidizer to be used can be a pyridine such as pyridine, dimethylaminopyridine or pyrrolidinopyridine, and is preferably dimethylaminopyridine.
  • the dicarboxylic anhydride to be used is not limited so long as it is the anhydride of an ⁇ , ⁇ -alkyl dicarboxylic acid having from 3 to 16 carbon atoms, and can preferably be succinic anhydride.
  • reaction temperature and the reaction time vary depending on the acid anhydride and deoxidizer used, in the case where succinic anhydride is used, and dimethylaminopyridine is used as the deoxidizer, the reaction is carried out at room temperature for 30 minutes.
  • the desired compound is collected from the reaction mixture according to ordinary methods. For example, after suitably neutralizing the reaction mixture and removing any insoluble matter, if present, by filtration, water and an immiscible organic solvent such as ethyl acetate are added, followed by washing with water, separating the organic layer containing the desired compound, drying the extract with anhydrous magnesium sulfate or the like, and distilling off the solvent to obtain the desired compound.
  • the resulting desired compound can be further purified by ordinary methods, for example, recrystallization, reprecipitation or chromatography if necessary.
  • the present step is a step, wherein active ester (18) is formed by reaction of the carboxyl group of compound (17) having a free carboxyl group with an ester-forming reagent in an inert solvent, and then reaction with a phenol which may be substituted.
  • the solvent to be used is not particularly limited so long as it does not inhibit the reaction, and it can be an aromatic hydrocarbon such as benzene, toluene or xylene; a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene or dichlorobenzene; an ester such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate or diethyl carbonate; a ketone such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone or cyclohexanone; a nitro compound such as nitroethane or nitrobenzene; a nitrile such as acetonitrile or isobutyronitrile; an amide such as formamide, dimethylformamide (DMF), dimethylacetamide or hexamethylphosphor
  • the phenol to be used is not particularly limited so long as it can be used as an active ester, and it can be 4-nitrophenol, 2,4-dinitrophenol, 2,4,5-trichlorophenol, 2,3,4,5,6-pentachlorophenol or 2,3,5,6-tetrafluorophenol, and is preferably pentachlorophenol.
  • the ester-forming reagent to be used can be, for example, an N-hydroxy compound such as N-hydroxysuccinimide, 1-hydroxybenzotriazole or N-hydroxy-5-norbornene-2,3-dicarboxyimide; a diimidazole compound such as 1,1′-oxalyldiimidazole or N,N′-carbonyldiimidazole; a disulfide compound such as 2,2′-dipyridyldisulfide; a succinic acid compound such as N,N′-disuccinimidylcarbonate; a phosphinic chloride compound such as N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride; an oxalate compound such as N,N′-disuccinimidyloxalate (DSO), N,N-diphthalimidyloxalate (DPO), N,N′-bis(norbornenylsuccinimi
  • reaction temperature and the reaction time vary depending on the ester-forming reagent and the kind of the solvent used, the reaction is carried out at from 0° C. to 100° C. for from 5 to 50 hours and, particularly in the case where pentachlorophenol and DCC are used in DMF, the reaction is carried out at room temperature for 18 hours.
  • the desired compound is collected from the reaction mixture according to ordinary methods. For example, after suitably neutralizing the reaction mixture and removing any insoluble matter, if present, by filtration, water and an immiscible organic solvent such as ethyl acetate are added, followed by washing with water, separating the organic layer containing the desired compound, drying the extract with anhydrous magnesium sulfate or the like, and distilling off the solvent to obtain the desired compound.
  • the resulting desired compound can be further purified by ordinary methods, for example, recrystallization, reprecipitation or chromatography if necessary.
  • the present step is a step, wherein high molecular weight derivative (20), which can be used as a carrier for oligonucleotide synthesis, is produced by allowing compound (18) having an activated carboxyl group obtained in Step E-3 to react with a high molecular weight substance (19), such as a control pore glass (CPG) bonded to an amino group, a hydroxyl group, a sulfhydryl group or the like through an alkylene group, in an inert solvent.
  • a high molecular weight substance (19) such as a control pore glass (CPG) bonded to an amino group, a hydroxyl group, a sulfhydryl group or the like through an alkylene group, in an inert solvent.
  • CPG control pore glass
  • the high molecular weight substance (19) used in the present step is not particularly limited so long as it is used as a carrier, but it is necessary to examine the particle size of the carrier, the size of surface area by a three-dimensional network structure, the ratio of hydrophilic group positions, the chemical composition, strength against pressure, and the like.
  • the carrier to be used can be a polysaccharide derivative such as cellulose, dextran or agarose; a synthetic polymer such as polyacrylamide gel, polystyrene resin or polyethylene glycol; or an inorganic substance such as silica gel, porous glass or a metal oxide.
  • a polysaccharide derivative such as cellulose, dextran or agarose
  • a synthetic polymer such as polyacrylamide gel, polystyrene resin or polyethylene glycol
  • an inorganic substance such as silica gel, porous glass or a metal oxide.
  • it can be a commercially available carrier such as aminopropyl-CPG, long chain aminoalkyl-CPG (these are manufactured by CPG Inc.), Cosmoseal NH 2 , Cosmoseal Diol (these are manufactured by Nacalai Tesque), CPC-Silica Carrier Silane Coated, aminopropyl-CPG-550 ⁇ , aminopropyl-CPG-1400 ⁇ , polyethylene glycol 5000 monomethyl ether (these are manufactured by Furuka Inc.), p-alkoxybenzyl alcohol resin, aminomethyl resin, hydroxymethyl resin (these are manufactured by Kokusan Kagaku Inc.) and polyethylene glycol 14000 monomethyl ether (these are manufactured by Union Carbide Inc.), but it is not limited to these.
  • aminopropyl-CPG long chain aminoalkyl-CPG
  • Cosmoseal NH 2 Cosmoseal Diol
  • CPC-Silica Carrier Silane Coated aminopropyl-CPG-550 ⁇
  • the functional group bonded to the carrier can preferably be an amino group, a sulfhydryl group, or a hydroxyl group.
  • the solvent used in the present step is not particularly limited so long as it does not inhibit the reaction and dissolves the starting material to a certain extent, and it can preferably be an aromatic hydrocarbon such as benzene, toluene or xylene; a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene or dichlorobenzene; an ester such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate or diethyl carbonate; a ketone such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone or cyclohexanone; a nitro compound such as nitroethane or nitrobenzene; a nitrile such as acetonitrile or isobutyronitrile; an amide such as formamide, dimethylformamide (DM
  • the reaction temperature is normally from ⁇ 20 to 150° C., preferably from 0 to 50° C.
  • the reaction time varies depending on the starting materials, the solvent, and the reaction temperature used, but it is normally from 1 to 200 hours, preferably from 24 to 100 hours.
  • the desired compound is collected from the reaction mixture according to ordinary methods. For example, the desired compound is obtained by recovering the high molecular weight carrier from the reaction mixture by filtration, washing with an organic solvent such as methylene chloride, and drying under reduced pressure.
  • the present step is a step, wherein 2-5A analog (1) is produced on a DNA automatic synthesizer by ordinary methods using the CPG (20) prepared in Step E-4, using the compounds (3), (8), (12) and (14) prepared in Step A-1, B-2, C-1 or D-1, and a commercially available phosphoramidite reagent (21).
  • the 2-5A analog having the desired nucleotide sequence can be synthesized according to a method described in the literature (Nucleic Acids Research, 12, 4539 (1984)), and the manual attached to the synthesizer, by a phosphoramidite method using a DNA synthesizer, for example, model 392 of Perkin Elmer Inc.
  • 5′-O-dimethoxytrityl-3′-O-(t-butyldimethylsilyl)-N-benzoyladenosine-2′-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) can be purchased from ChemGene Inc. (catalogue No.: ANP-5681).
  • the amidite reagent for the compounds (3), (8), (12), (14) and (21) is activated using an acid catalyst to form a phosphorous acid tri-ester bond, and it is oxidized to a phosphoric acid tri-ester using an appropriate oxidizing agent, or it is made into a thiophosphoric tri-ester using an appropriate thioating agent.
  • the acidic substance used as a catalyst in the condensation reaction of the present step can be an acidic substance such as a tetrazole, and is preferably tetrazole or ethylthiotetrazole.
  • the oxidizing agent used in the oxidation reaction of the present step is not particularly limited so long as it is normally used in oxidation reactions, and is preferably an inorganic metal oxidizing agent such as a manganese oxide, i.e., potassium permanganate or manganese dioxide; a ruthenium oxide, i.e., ruthenium tetraoxide; a selenium compound, i.e., selenium dioxide; an iron compound, i.e., iron chloride; an osmium compound, i.e., osmium tetraoxide; a silver compound, i.e., silver oxide; a mercury compound, i.e., mercury acetate; a lead oxide compound, i.
  • the solvent to be used is not particularly limited so long as it does not inhibit the reaction and dissolves the starting material to a certain extent, and it can preferably be an aromatic hydrocarbon such as benzene, toluene or xylene; a halogenated hydrocarbon such as methylene chloride or chloroform; an ether such as ether, tetrahydrofuran, dioxane or dimethoxyethane; an amide such as dimethylformamide, dimethylacetamide or hexamethylphosphortriamide; a sulfoxide such as dimethyl sulfoxide; an alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol or isoamyl alcohol; a dilute acid such as aqueous sulfuric acid; a dilute base such as aqueous sodium hydroxide; water; a ketone such as acetone or methyl ethyl ketone
  • the thioate derivative can be obtained according to a method described in the literature (Tetrahedron Letters, 32, 3005 (1991), J. Am Chem. Soc., 112, 1253 (1990)) using a reagent such as sulphur, tetraethyl thiuram disulfide (TETD, Applied Biosystems Inc., or Beaucage reagent (Millipore Inc.) for forming a thioate by reacting with a phosphite.
  • a reagent such as sulphur, tetraethyl thiuram disulfide (TETD, Applied Biosystems Inc., or Beaucage reagent (Millipore Inc.)
  • the reaction temperature is normally from 0 to 150° C., preferably from 10 to 60° C.
  • the reaction time varies depending on the starting materials, the solvent and the reaction temperature used, but it is normally from 1 minute to 20 hours, preferably from 1 minute to 1 hour.
  • the H-phosphonic acid compound (4) or (9) obtained in Step A-2 or B-3 is condensed to form a phosphoric tri-ester bond in the present step, after it is condensed, for example, in the presence of a condensing agent such as pivaloyl chloride and a deoxidizer to form the H-phosphonic acid diester bond, the H-phosphonic acid bond can be converted to the phosphoric acid diester bond using an oxidizing agent.
  • a condensing agent such as pivaloyl chloride and a deoxidizer
  • the solvent used in the present step is not particularly limited so long as it does not inhibit the reaction, but anhydrous acetonitrile is preferably used.
  • anhydrous acetonitrile is preferably used as the reagent used as the condensing agent.
  • an acid chloride of a carboxylic acid or phosphoric acid is used, and pivaloyl chloride is preferably used.
  • the oxidizing agent for oxidizing the ODN having a H-phosphonic acid bond to a phosphodiester type ODN is not particularly limited so long as it is normally used for oxidation reactions, and can be a inorganic metal oxidizing agent such as a manganese oxide, e.g., potassium permanganate or manganese dioxide; a ruthenium oxide, e.g., ruthenium tetraoxide; a selenium compound, e.g., selenium dioxide; an iron compound, e.g., iron chloride; an osmium compound, e.g., osmium tetraoxide; a silver compound, e.g., silver oxide; a mercury compound, e.g., mercury acetate; a lead oxide compound, e.g., lead oxide or lead tetraoxide; a chromic acid compound, e.g., potassium chromate, a chromic acid-sulfuric acid
  • the deoxidizer to be used can be a heterocyclic amine such as pyridine or dimethylaminopyridine; or an aliphatic amine such as trimethylamine, triethylamine or diisopropylethylamine; and is preferably an aliphatic amine (particularly diisopropylethylamine).
  • the reaction temperature is not particularly limited but it is normally from ⁇ 50 to 50° C., preferably room temperature.
  • the reaction time varies depending on the starting materials, the reagent and the temperature used, but it is normally from 5 minutes to 30 hours, preferably in the case where the reaction is carried out at room temperature, it is 30 minutes.
  • the solvent in the reaction for forming a methoxyethylamino phosphate group is not particularly limited so long as it does not inhibit the reaction, but carbon tetrachloride that is normally used as a reagent is used at a solvent amount.
  • the reaction temperature is not particularly limited in a range of from ⁇ 50 to 100° C., but in the case where the reaction is carried out at room temperature, the reaction time is from 1 to 10 hours.
  • the solvent used in the present step is not particularly limited so long as it does not inhibit the reaction, but an aromatic amine such as pyridine is preferably used.
  • the condensing agent used in the condensation can be dicyclocarbodiimide (DCC), mesitylenesulfonic chloride (Ms-Cl), triisopropylbenzenesulfonic chloride, mesitylenesulfonic acid triazolide (MST), mesitylenesulfonic acid-3-nitrotriazolide (MSNT), triisopropylbenzenesulfonic acid tetrazolide (TPS-Te), triisopropylbenzenesulfonic acid nitroimidazolide (TPS-NI) or triisopropylbenzenesulfonic acid pyridyltetrazolide, and is preferably MSNT, TPS-Te and TPS-NI.
  • DCC dicyclocarbodiimide
  • MST mesitylenesulfonic acid triazolide
  • MSNT mesitylenesulfonic acid-3-nitrotriazolide
  • TPS-Te triisopropy
  • the reaction temperature is not particularly limited in a range of from ⁇ 10 to 100° C., but the reaction is normally carried out at room temperature.
  • the reaction time varies depending on the solvent used and the reaction temperature, but in the case where pyridine is used as the reaction solvent, and the reaction is carried out at room temperature, it is 30 minutes.
  • the resulting crude 2-5A analog can be confirmed by purification using a reverse phase chromatocolumn and analyzing the purity of the purified product by HPLC.
  • the chain length of the thus obtained oligonucleotide analog is normally from 2 to 50, preferably from 10 to 30 nucleoside units.
  • the present step is to prepare 2-5A analog (1) on a DNA automatic synthesizer by ordinary methods using CPG (22), using the compounds (3), (4), (5), (8), (9), (10), (12) or (14) prepared in Step A-1, A-2, A-3, B-2, B-3, B-4, C-1 or D-1 and (21).
  • CPG (22) is the same as the compound (24) described in Process G of Japanese Patent Application (Kokai) No. 2002-249497, and the present step is carried out similarly to Step F-1.
  • the present step is a step, wherein 2-5A analog (1) is produced on a DNA automatic synthesizer by ordinary methods using CPG (23), using the compounds (3), (4), (5), (8), (9), (10), (12) or (14) prepared in Step A-1, A-2, A-3, B-2, B-3, B-4, C-1 or D-1 and (21).
  • CPG (23) is the same as the compound (4) described in Japanese Patent Application (Kokai) No. Hei 7-53587, and the present step is carried out similarly to Step F-1.
  • the halogen can be, for example, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and is preferably a chlorine atom, a bromine atom, or an iodine atom.
  • the compound having a halide group to be used is not particularly limited so long as it is a compound having a halide group which can be reacted with a thiophosphoric acid group, and can be, for example, an “alkyl halide which may be substituted” such as an ethyl halide, a propyl halide, a butyl halide, a 2-halo ethanol, a 3-halo propanol, or a 4-halo butanol; an “acyloxyalkyl halide” such as a 2-(stearoyloxy)ethyl halide, a 2-(myristoyloxy)ethyl halide, a 2-(decanoyloxy)ethyl halide, a 2-(benzoyloxy)ethyl halide, a 2-(pivaloyloxy)ethyl halide, a 2-(2,2-dimethyloctadecanoyloxy
  • a 2-stearoyloxyethyl halide and a 2-(2,2-dimethyloctadecanoyloxy)ethyl halide are preferred.
  • the base to be used can be a heterocyclic amine such as pyridine or dimethylaminopyridine; or an aliphatic amine such as trimethylamine, triethylamine or diisopropylamine; and is preferably a heterocyclic amine (particularly pyridine).
  • a heterocyclic amine such as pyridine or dimethylaminopyridine
  • an aliphatic amine such as trimethylamine, triethylamine or diisopropylamine
  • the solvent to be used there is no particular limitation on the solvent to be used, provided that it does not inhibit the reaction and dissolves the starting material to a certain extent, and it can be water; an amide such as dimethylformamide, dimethylacetamide or hexamethylphosphortriamide; a sulfoxide such as dimethyl sulfoxide; a heterocyclic amine such as pyridine; a nitrile such as acetonitrile; or a mixture of these solvents; and is preferably dimethylformamide.
  • an amide such as dimethylformamide, dimethylacetamide or hexamethylphosphortriamide
  • a sulfoxide such as dimethyl sulfoxide
  • a heterocyclic amine such as pyridine
  • a nitrile such as acetonitrile
  • a mixture of these solvents and is preferably dimethylformamide.
  • the reaction temperature is not particularly limited in a range of from ⁇ 50 to 100° C., but the reaction is normally carried out at room temperature.
  • the reaction time varies depending on the material, the reagent used, and the temperature, but it is normally from 10 hours to 100 hours.
  • reaction speed can also be appropriately increased by adding an iodide salt such as tetrabutylammonium iodide.
  • an iodide salt such as tetrabutylammonium iodide.
  • a 2-5A antisense oligonucleotide can be synthesized by condensing a phosphoramidite serving as a linker, such as DMT-butanol-CED phosphoramidite (ChemGene) or Spacer phosphoramidite 18 (GlenResearch), to CPG to which is bonded an oligonucleotide having the desired antisense sequence that is protected with a protecting group, followed by carrying out the procedure of the present step.
  • a phosphoramidite serving as a linker
  • a phosphoramidite serving as a linker
  • DMT-butanol-CED phosphoramidite ChemGene
  • Spacer phosphoramidite 18 Spacer phosphoramidite 18
  • a modified oligonucleotide in which the oxygen atom at the 2′ position of the sugar portion is bridged to a carbon atom at the 4′ position with an alkylene group according to the method described in Japanese Patent Application (Kokai) No. Hei 10-304889 or Japanese Patent Application (Kokai) No. 2000-297097.
  • a modified oligonucleotide having a 2′-O-methoxyethoxy group can be synthesized by referring to the literature (Teplove, M. et al., Nat. Struct. Biol.
  • the antitumor activity (cytocidal activity) of the present compounds can be investigated by adding the present compounds to cancer cells in a medium, and culturing the cells, followed by counting the number of viable cells using the MTT assay method (Tim Mosmann, J. Immunological Methods, 1983: 65, 55-63), the MTS assay method (Rotter, B. A., Thompson, B. K., Clarkin, S., Owen, T. C. Nat. Toxins 1993; 1(5): 303-7), the XTT assay method (Meshulam, T., Levitz, S. M., Christin, L., Diamond, R. D. J. Infect. Dis. 1995; 172(4): 1153-6), or Trypan blue staining.
  • Compounds of the invention will show anti-cancer activity against any type of malignant neoplasm and leukemia that express Rnase L, a target protein of this invention, including lung cancer, colorectal cancer, breast cancer, renal cancer, melanoma and glioma.
  • the antivirus activity of the present compounds can be investigated using an infected cell culture system such as HeLa cells, MDCK cells, MRC-5 cells or the like, by adding the present compounds to virus cells, such as of vaccinia virus, influenza virus or cytomegalovirus, in a medium either before or after infection, culturing for a predetermined amount of time, and then measuring the virus growth inhibition rate using the plaque assay method which measures virus infection titer (Kobayashi, N., Nagata, K. Virus Experimental Protocols, Medical View Publishing), or the ELISA method which measures the level of virus antigen (Okuno, Y., Tanaka, K., Baba, K., Maeda, A., Kunita, N., Ueda, J. Clin. Microbiol., Jun. 1, 1990; 28(6): 1308-13).
  • the present compounds have antiviral activity to the aforesaid viruses and also to hepatitis C.
  • the administration forms of the 2-5A analogs of general formula (1) of the present invention can include, for example, oral administration by tablets, capsules, granules, powders or syrups, or parenteral administration by injection or suppositories.
  • pharmaceutically acceptable carriers such as additives such as excipients (which include, for example, organic excipients such as sugar derivatives, e.g., lactose, sucrose, glucose, mannitol and sorbitol; starch derivatives, e.g., corn starch, potato starch, ⁇ -starch and dextrin; cellulose derivatives, e.g., crystalline cellulose; gum arabic; dextran; and pullulan; and inorganic excipients such as silicate derivatives, e.g., light silicic anhydride, synthetic aluminum silicate, calcium silicate and magnesium aluminate meta-silicate; phosphates, e.g., calcium hydrogenphosphate; carbonates
  • excipients
  • the amount of the 2-5A analog of the present invention used varies depending on the symptoms, the age, the administration method, and the like, it is desirable to administer to the patient, such as a mammal, e.g., a human, once to several times a day, and, in the case of oral administration, 0.01 mg/kg body weight (preferably 0.1 mg/kg body weight) per time as a lower limit and 1000 mg/kg body weight (preferably 100 mg/kg body weight) as an upper limit, and, in the case of intravenous administration, 0.001 mg/kg body weight (preferably 0.01 mg/kg body weight) per time as a lower limit and 100 mg/kg body weight (preferably 10 mg/kg body weight) as an upper limit corresponding to the symptoms of the patient.
  • a mammal e.g., a human
  • Topical administration e.g., pulmonary, intratracheal and intranasal
  • parenteral administration modes including intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration.
  • preparations may be used in combination with other antitumor agents, for example, nitrosourea type chemicals such as 5FU, AraC, ACNU or BCNU, cisplatin, daunomycin, adriamycin, mitomycin C, vincristine, and taxol.
  • nitrosourea type chemicals such as 5FU, AraC, ACNU or BCNU
  • cisplatin adriamycin
  • mitomycin C vincristine
  • taxol taxol
  • the ABI Model 392 DNA/RNA Synthesizer (Applied Biosystems) was used as the DNA synthesizer.
  • the solvents, reagents, and phosphoramidite concentrations in each synthesis cycle were the same as in the case of general natural oligonucleotide synthesis, and the products of Applied Biosystems were used for those reagents and solvents other than the phosphoramidite and sulfurizing agent.
  • Synthesis was carried out using the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 ⁇ mol of RNA.
  • 3′-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite-(ChemGene) was used as the phosphoramidite in cycles 1 and 2, while the compound of Example 8a described in Japanese Patent Application (Kokai) No. Hei 11-246592 was used in cycle 3.
  • xanthane hydride Tokyo Kasei Kogyo
  • iodine was used in cycle 3.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30° C. to remove the silyl group.
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 0-13% CH 3 CN (linear gradient, 30 min.); 40° C.; 10 ml/min; 254 nm), and the fractions that eluted at 20.9, 22.7, 25.4 and 28.0 minutes corresponding to the four diastereomers were collected.
  • reverse phase HPLC Shiadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)
  • TEAA triethylamine acetate
  • pH 7 pH 7
  • CH 3 CN linear gradient, 30 min.
  • 40° C. 10 ml/min; 254 nm
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30° C. to remove the silyl group.
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)); 0.1 M aqueous triethyl amine acetate (TEAA), pH 7; 0-15% CH 3 CN (linear gradient, 30 min.); 40° C.; 10 ml/min; 254 nm), and the fraction that eluted at 16.7 minutes was collected.
  • reverse phase HPLC Shiadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)
  • TEAA triethyl amine acetate
  • pH 7 pH 7
  • CH 3 CN linear gradient, 30 min.
  • 40° C. 10 ml/min; 254 nm
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1).
  • the solvent was then distilled off under reduced pressure and the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 60% CH 3 CN (isocratic); 40° C.; 10 ml/min; 254 nm), and the fractions that eluted at 9.5 and 11.8 minutes as diastereomers were collected. After the solvent was distilled off under reduced pressure, 80% aqueous acetic acid was added thereto, the mixture was left to stand for 30 minutes, and the DMTr group was removed.
  • reverse phase HPLC Shiadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)
  • TEAA triethylamine acetate
  • pH 7 pH 7
  • 60% CH 3 CN isocratic
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 0-15% CH 3 CN (linear gradient, 30 min.); 40° C.; 10 ml/min; 254 nm), and the fractions that eluted at 16.5-19.1 minutes were collected.
  • reverse phase HPLC Shiadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)
  • TEAA triethylamine acetate
  • pH 7 pH 7
  • CH 3 CN linear gradient, 30 min.
  • 40° C. 10 ml/min; 254 nm
  • Synthesis was carried out using Bz-Adenosine-RNA 500 (Glen Research Co.) (2.0 mol) as the 5′-O-DMTr-riboadenosine analog bound to a CPG support with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 ⁇ mol of RNA.
  • 3′-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1 and 2 and Chemical Phosphorylation Reagent II (Glen Research Co.) was used in cycle 3.
  • Xanthane hydride Tokyo Kasei Kogyo was used as the sulfurizing agent in cycles 1, 2 and 3.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30° C. to remove the silyl group.
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 6-25% CH 3 CN (linear gradient, 30 min.); 40° C.; 10 ml/min; 254 nm), and the four fractions that eluted at 13.3, 13.7, 13.9 and 14.4 minutes corresponding to the four diastereomers were collected.
  • reverse phase HPLC Shiadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)
  • TEAA triethylamine acetate
  • pH 7 pH 7
  • 6-25% CH 3 CN linear gradient, 30 min.
  • 40° C. 10 ml/min; 254 nm
  • Example 2 compound 30 nmol of Example 2 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 ⁇ l of pivaloyloxymethyl chloride (Tokyo Kasei Kogyo), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 50 ⁇ l of AcOEt.
  • Tokyo Kasei Kogyo pivaloyloxymethyl chloride
  • Tokyo Kasei Kogyo tetrabutylammonium iodide
  • triethylamine 1 ⁇ l of triethylamine
  • Example 2 compound 30 nmol of Example 2 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 ⁇ l of thioacetic acid S-(2-bromo-ethyl) ester (Bauer, L. et al. J. Org. Chem. 1965, 30, 949-951), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 50 ⁇ l of AcOEt.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30° C. to remove the silyl group.
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 20 min.); 40° C.; 10 ml/min; 254 nm), and the fractions that eluted at 12.1 and 13.0 minutes corresponding to the two diastereomers were collected.
  • reverse phase HPLC Shiadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)
  • TEAA triethylamine acetate
  • pH 7 pH 7
  • 9-25% CH 3 CN linear gradient, 20 min.
  • 40° C. 10 ml/min; 254 nm
  • the present compound eluted in the vicinity of 8.66 and 8.98 minutes when analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 ⁇ 50 mm)); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 0-15% CH 3 CN (linear gradient, 10 min); 60° C.; 1 ml/min). Yield: 768 nmol as UV measured value at 260 nm, ⁇ max (H 2 O) 258 nm, ESI-Mass (negative): 1090.2 [M-H] ⁇ .
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30° C. to remove the silyl group.
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 20 min.); 40° C.; 10 ml/min; 254 nm), and the fractions that eluted at 11.5 and 12.4 minutes corresponding to the two diastereomers were collected.
  • reverse phase HPLC Shiadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)
  • TEAA triethylamine acetate
  • pH 7 pH 7
  • 9-25% CH 3 CN linear gradient, 20 min.
  • 40° C. 10 ml/min; 254 nm
  • Example 7 compound 30 nmol of Example 7 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 ⁇ l of pivaloyloxymethyl chloride (Tokyo Kasei Kogyo), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 50 ⁇ l of AcOEt.
  • Tokyo Kasei Kogyo pivaloyloxymethyl chloride
  • Tokyo Kasei Kogyo tetrabutylammonium iodide
  • triethylamine 1 ⁇ l of triethylamine
  • Example 8 compound 30 nmol of Example 8 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 ⁇ l of pivaloyloxymethyl chloride (Tokyo Kasei Kogyo), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 50 ⁇ l of AcOEt.
  • Tokyo Kasei Kogyo pivaloyloxymethyl chloride
  • Tokyo Kasei Kogyo tetrabutylammonium iodide
  • triethylamine 1 ⁇ l of triethylamine
  • Example 7 compound 30 nmol of Example 7 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 ⁇ l of thioacetic acid S-(2-bromo-ethyl) ester (Bauer, L. et al. J. Org. Chem. 1965, 30, 949-951), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 50 ⁇ l of AcOEt.
  • Example 8 compound 30 nmol of Example 8 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 ⁇ l of thioacetic acid S-(2-bromo-ethyl) ester (Bauer, L. et al. J. Org. Chem. 1965, 30, 949-951), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 50 ⁇ l of AcOEt.
  • Example 2 compound 30 nmol of Example 2 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 ⁇ l of 2-(pivaloyloxy)ethyl bromide (Preparation process described in EP0395313), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 50 ⁇ l of AcOEt.
  • Example 2 compound 30 nmol of Example 2 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 ⁇ l of 2-(benzoyloxy)ethyl bromide (Tokyo Kasei Kogyo), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 50 ⁇ l of AcOEt.
  • 2-(benzoyloxy)ethyl bromide Tokyo Kasei Kogyo
  • Example 2 compound 30 nmol of Example 2 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 50 ⁇ l of AcOEt.
  • Example 2 compound 30 nmol of Example 2 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 mg of 2-(myristoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 50 ⁇ l of AcOEt.
  • Example 2 compound 30 nmol of Example 2 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 mg of 2-(decanoyloxy)ethyl bromide (Devinsky, Gustav et al., Collect. Czech. Chem. Commun. 49, 12, 1984, 2819-2827), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 50 ⁇ l of AcOEt.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure, and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue, followed by reacting for 5 hours at 30° C. to remove the DMTr group and the silyl group.
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 5-17% CH 3 CN (linear gradient, 20 min.); 40° C.; 10 ml/min; 254 nm), and the fraction that eluted at 14.9 minutes was collected.
  • reverse phase HPLC Shiadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)
  • TEAA triethylamine acetate
  • pH 7 pH 7
  • 5-17% CH 3 CN linear gradient, 20 min.
  • 40° C. 10 ml/min; 254 nm
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure, and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue, followed by reacting for 5 hours at 30° C. to remove the DMTr group and the silyl group.
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)); 0.1 M aqueous triethylamine acetate. (TEAA), pH 7; 5-17% CH 3 CN (linear gradient, 20 min.); 40° C.; 10 ml/min; 254 nm), and the fraction that eluted at 15.5 minutes was collected.
  • TEAA reverse phase HPLC
  • pH 7 pH 7
  • CH 3 CN linear gradient, 20 min.
  • 40° C. 10 ml/min; 254 nm
  • Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 ⁇ mol of RNA. 31-Phosphate CPG (Glen Research) (1.0 ⁇ mol) was used as the solid phase carrier.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure, and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue, followed by reacting for 5 hours at 30° C. to remove the DMTr group and the silyl group.
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 ⁇ 50 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 2.5-10% CH 3 CN (linear gradient, 10 min.); 60° C.; 2 ml/min), and the fraction that eluted at 4.8 minutes was collected.
  • Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 ⁇ mol of RNA.
  • 3′-Phosphate CPG (Glen Research) (1.0 ⁇ mol) was used as the solid phase carrier.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure, and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue, followed by reacting for 5 hours at 30° C. to remove the DMTr group and the silyl group.
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 ⁇ 50 mm)); 0.1 M aqueous triethyl amine acetate (TEAA), pH 7; 5-10% CH 3 CN (linear gradient, 10 min.); 60° C.; 2 ml/min), and the fractions that eluted at 6.0 and 6.4 minutes were collected.
  • reverse phase HPLC Shiadzu Seisakusho LC-VP; column (Merck chromolith (4.6 ⁇ 50 mm)
  • TEAA triethyl amine acetate
  • pH 7 pH 7
  • 5-10% CH 3 CN linear gradient, 10 min.
  • 60° C. 60° C.; 2 ml/min
  • reaction mixture was purified using a silica gel column (elution by hexane-ethyl acetate (7:1) solvent mixture) to obtain 230 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromide to be used below.
  • Example 19 compound 100 nmol of Example 19 compound were dissolved in 100 ⁇ l of anhydrous DMF, and 3 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromide, and 3 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 19 compound 100 nmol of Example 19 compound were dissolved in 100 ⁇ l of anhydrous DMF, and 3 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 3 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 19 compound 100 nmol of Example 19 compound were dissolved in 100 ⁇ l of anhydrous DMF, and 3 mg of 2-(myristoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 3 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 19 compound 100 nmol of Example 19 compound were dissolved in 100 ⁇ l of anhydrous DMF, and 3 mg of 2-(decanoyloxy)ethyl bromide (Devinsky, Gustav et al., Collect. Czech. Chem. Commun. 49, 12, 1984, 2819-2827), and 3 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 20 compound 30 nmol of Example 20 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 100 ⁇ l of AcOEt.
  • Example 21 compound were dissolved in 30 ⁇ l of anhydrous DMF, and 1 mg of 2-(stearoyloxy) ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 ⁇ l of water were added, and the aqueous layer was washed three times with 100 ⁇ l of AcOEt.
  • Example 18 compound 100 nmol of Example 18 compound were dissolved in 100 ⁇ l of anhydrous DMF, and 3 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 3 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 18 compound 100 nmol of Example 18 compound were dissolved in 100 ⁇ l of anhydrous DMF, and 3 mg of 2-(myristoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 3 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 18 compound 100 nmol of Example 18 compound were dissolved in 100 ⁇ l of anhydrous DMF, and 3 mg of 2-(decanoyloxy)ethyl bromide (Devinsky, Gustav et al., Collect. Czech. Chem. Commun. 49, 12, 1984, 2819-2827), and 3 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • cycle 2 Xanthane hydride (0.02 M)/acetonitrile-pyridine (9:1 mixed solvent); 15 min.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30° C. to remove the silyl group.
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 30 min.); 40° C.; 10 ml/min; 254 nm), and the fractions that eluted at 10.9 and 12.0 minutes corresponding to the two diastereomer were collected.
  • reverse phase HPLC Shiadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)
  • TEAA triethylamine acetate
  • pH 7 pH 7
  • 9-25% CH 3 CN linear gradient, 30 min.
  • 40° C. 10 ml/min; 254 nm
  • cycle 2 Xanthane hydride (0.02 M)/acetonitrile-pyridine (9:1 mixed solvent); 15 min.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30° C. to remove the silyl group.
  • the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 30 min.); 40° C.; 10 ml/min; 254 nm), and the fractions that eluted at 11.5 and 12.7 minutes corresponding to the two diastereomers were collected.
  • reverse phase HPLC Shiadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 ⁇ 250 mm)
  • TEAA triethylamine acetate
  • pH 7 pH 7
  • 9-25% CH 3 CN linear gradient, 30 min.
  • 40° C. 10 ml/min; 254 nm
  • Example 2 compound 100 nmol of Example 2 compound were dissolved in 50 ⁇ l of anhydrous DMF, and 2 ⁇ l of 2-bromoethanol (Tokyo Kasei Kogyo), and 2 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 200 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 19 compound 100 nmol of Example 19 compound were dissolved in 50 ⁇ l of anhydrous DMF, and 2 ⁇ l of 2-bromoethanol (Tokyo Kasei Kogyo), and 2 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 200 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Oxidation (cycle 4) Iodine/water/pyridine/tetrahydrofuran; 15 sec.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1).
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1).
  • Example 35 compound 40 nmol of Example 35 compound were dissolved in 40 ⁇ l of anhydrous DMF, and 1 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromides described in Example 22, and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 36 compound 40 nmol of Example 36 compound were dissolved in 40 ⁇ l of anhydrous DMF, and 1 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromide described in Example 22, and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 35 compound 40 nmol of Example 35 compound were dissolved in 40 ⁇ l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 36 compound 40 nmol of Example 36 compound were dissolved in 40 ⁇ l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • 3420984 was used as the phosphoramidite in cycle 1
  • 3′-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used in cycle 2
  • 5′-DMT-3′-(O-methyl) adenosine(N-bz)2′-phosphoramidite (ChemGene) was used in cycle 3
  • the compound of Example 8a described in Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4.
  • xanthane hydride Tokyo Kasei Kogyo
  • iodine was used in cycle 4.
  • Oxidation (cycle 4) Iodine/water/pyridine/tetrahydrofuran; 15 sec.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure, and the pH was accurately adjusted to 2.0 by adding 1 ml of aqueous hydrochloric acid (0.01 N) to the remaining residue, followed by reacting for 5 hours at 30° C. to remove the DMTr group and the silyl group.
  • 3420984 was used as the phosphoramidite in cycle 1
  • 3′-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used in cycle 2
  • 5′-DMT-3′-(O-methyl) adenosine(N-bz)2′-phosphoramidite (ChemGene) was used in cycle 3
  • the compound of Example 8a described in Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4.
  • xanthane hydride Tokyo Kasei Kogyo
  • iodine was used in cycles 3 and 4.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1).
  • Example 41 compound 40 nmol of Example 41 compound were dissolved in 40 ⁇ l of anhydrous DMF, and 1 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromide described in Example 22, and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 42 compound 40 nmol of Example 42 compound were dissolved in 40 ⁇ l of anhydrous DMF, and 1 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromide described in Example 22, and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 41 compound 80 nmol of Example 41 compound were dissolved in 100 ⁇ l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 ⁇ l of pyridine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 500 ⁇ l of water were added, and the aqueous layer was washed three times with 500 ⁇ l of AcOEt.
  • Example 42 compound 80 nmol of Example 42 compound were dissolved in 100 ⁇ l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 ⁇ l of pyridine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 500 ⁇ l of water were added, and the aqueous layer was washed three times with 500 ⁇ l of AcOEt.
  • Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 ⁇ mol of RNA.
  • 3′-Phosphate CPG (Glen Research) (0.5 ⁇ mol) was used as the solid phase carrier.
  • 5′-DMT-3′-(O-methyl) Adenosine(N-bz)2′-phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1 and 3
  • 3′-tBDSilyl-riboAdenosine (N-bz) phosphoramidite (ChemGene) was used in cycle 2
  • the compound of Example 8a described in Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4.
  • xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 1, 2 and 3, and iodine was used in cycle 4.
  • Oxidation (cycle 4) Iodine/water/pyridine/tetrahydrofuran; 15 sec.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1).
  • Example 47 compound 40 nmol of Example 47 compound were dissolved in 40 ⁇ l of anhydrous DMF, and 1 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromide described in Example 22, and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 47 compound 40 nmol of Example 47 compound were dissolved in 40 ⁇ l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 ⁇ l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 ⁇ l of water were added, and the aqueous layer was washed three times with 200 ⁇ l of AcOEt.
  • Example 50 Compound (Exemplary Compound No. 1929) HOC 2 H 4 O—P( ⁇ O) (OH)—K 2-1 —P( ⁇ O)(OH)—K 1-1 —P( ⁇ O)(OH)—K 2-1 —P( ⁇ O)(OH)—L 1 —P( ⁇ O)(OH)—L 1 -p-G e -p-A e -p-G e -p-A e -p-C e -p-C n -p-C n -p-T n -p-G n -p-A n -p-A n -p-C n -p-A n -p-G n -p-T n -p-G e -p-A e -p-T n -p-G e -p-A e -p-T e -p-C e -hp
  • a 2-5A analog having the desired sequence was synthesized by coupling various phosphoramidites in order based on one condensation cycle consisting of the following steps 1) to 4) using a DNA synthesizer, a synthesis program for ordinary synthesis of 1 ⁇ mol of RNA, and 1 ⁇ mol of the compound described in Example 12b of Patent Application (Kokai) No. Hei 7-87982 as the solid phase support.
  • adenine (dA bz ) phosphoramidite, guanine (dG ibu ) phosphoramidite, cytosine (dC bz ) phosphoramidite, and thymine (T) phosphoramidite were used for the sequences equivalent to natural type nucleotides, while the compounds of Examples 14, 27, 22 and 9 described in Japanese Patent No. 3420984 were used for the sequences equivalent to non-natural type nucleotides (A e , G e , C e , T e ).
  • DMT-butanol-CED phosphoramidite (ChemGene) was used for the phosphoramidite equivalent to L 1 , and 5′-DMT-3′-(O-methyl)adenosine(N-bz)2′-phosphoramidite (ChemGene), 3′-tBDsilyl-riboadenosine(N-bz)phosphoramidite (ChemGene), 5′-DMT-3′-(O-methyl)adenosine(N-bz)2′-phosphoramidite (ChemGene), and the phosphoramidite of Example 8a described in Patent Application (Kokai) No. Hei 11-246592, were coupled in order.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the protecting group on the nucleic acid base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1). The solvent was then distilled off under reduced pressure, and 1 ml of triethylamine trihydrofluoride was added to the residue followed by stirring at room temperature.
  • the band that absorbed UV in the gel was cut out and eluted from the gel with 1 ml of an elution buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA (pH 8.0), 0.1% SDS). The remaining gel was filtered off, 4 ml of EtOH were added to the filtrate, which was then allowed to stand for 1 hour at ⁇ 20° C. followed by centrifugation to obtain a pellet-like precipitate.
  • an elution buffer 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA (pH 8.0), 0.1% SDS.
  • Example 51 Compound (Exemplary Compound No. 1930) HOC 2 H 4 O—P( ⁇ O)(OH)—K 2-1 —P( ⁇ O)(OH)—K 1-1 —P( ⁇ O)(OH)—K 2-1 —P( ⁇ O)(OH)—L 1 —P( ⁇ O)(OH)—L 1 -p-T e -p-C e -p-T e -p-T e -p-G n -p-T n -p-T n -p-T n -p-G n -p-T n -p-A n -p-A n -p-G n -p-A n p-G n -p-A n p-G n -p-A n p-G n -p-A n p-G n -p-A n p-G n -p-A n -
  • the title compound was obtained according to a similar method to Example 50.
  • Example 52 Compound (Exemplary compound No. 1931) HOC 2 H 4 O—P( ⁇ O)(OH)—K 2-1 —P( ⁇ O)(OH)—K 1-1 —P( ⁇ O)(OH)—K 2-1 —P( ⁇ O)(OH)—L 1 —P( ⁇ O)(OH)—L 1 -p-T e -p-T e -p-C e -p-A e -p-G e -p-G n -p-C n -p-C n -p-T n -p-C n -p-C n -p-C n -p-A n -p-T n -p-A n -p-T n -p-A n -p-T n -p-p-T n -p-G e -p-G e -p-A e -p-A e -
  • the title compound was obtained according to a similar method to Example 50.
  • the title compound was obtained according to a similar method to Example 50.
  • the present compound was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 ⁇ 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 8-12% CH 3 CN (linear gradient, 10 min); 60° C.; 2 ml/min), and the fraction at 8.0-10.0 minutes was collected.
  • the title compound was obtained according to a similar method to Example 50.
  • the title compound was obtained according to a similar method to Example 50.
  • Example 56 Compound (Exemplary Compound No. 1935) HOC 2 H 4 O—P( ⁇ O)(OH)—K 2-1 —P( ⁇ O)(OH)—K 1-1 —P( ⁇ O)(OH)—K 2-1 —P( ⁇ O)(OH)—L 1 —P( ⁇ O)(OH)—L 1 -p-C e -p-A e -p-G e -p-C e -p-C e -p-A n -p-T n -p-G n -p-G n -p-T n -p-C n -p-C n -p-C n -p-C n -p-C n -p-C n -p-C n -p-C n -p-C n -p-C n -p-C n -p-C n -p-C n -p-
  • the title compound was obtained according to a similar method to Example 50.
  • the title compound was obtained according to a similar method to Example 50.
  • Spacer phosphoramidite 18 (Glen Research Inc.) was used as the phosphoramidite corresponding to L 2 .
  • the present compound was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 ⁇ 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 8-12% CH 3 CN (linear gradient, 10 min); 60° C.; 2 ml/min), and the fraction at 8.0-10.0 minutes was collected.
  • Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 ⁇ mol of RNA.
  • 3′-Phosphate CPG (Glen Research) (0.2 mol) was used as the solid phase carrier.
  • 3′-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1, 2 and 3, and the compound of Example 8a described in Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4.
  • xanthane hydride Tokyo Kasei Kogyo
  • iodine was used in cycle 4.
  • Oxidation (cycle 4) Iodine/water/pyridine/tetrahydrofuran; 15 sec.
  • the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol (3:1).
  • aqueous hydrochloric acid (0.01N) was added to the remaining residue to accurately adjust the pH to 2.0, followed by reaction at 30° C. for 5 hours to remove the DMTr group and the silyl group. After neutralization with aqueous ammonia, the deprotected silanol and DMTrOH were removed by extraction with ethyl acetate.
  • Example 58 compound 40 nmol of Example 58 compound were dissolved in 100 ⁇ l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 ⁇ l of pyridine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 500 ⁇ l of water were added, and the aqueous layer was washed three times with 500 ⁇ l of AcOEt.
  • Human lung cancer cell line A549 cells were plated at a density of 800 cells/200 ⁇ l in a 96-well plate using RPMI1640 (Gibco BRL) (containing 10% Fetal Bovine Serum (Hyclone)) for the medium followed by culturing overnight in 5% CO 2 at 37° C. Each 2-5A analog was added to each well so that the final concentration became 10 ⁇ M, followed by culturing for 72 hours (3 days).
  • MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
  • MTT/RPMI1640 concentration 5 mg/ml
  • UV absorbance at 540 nm was measured to determine the relative ratio of the number of viable cells of the compound dose group to the number of viable cells of an untreated cell group at 72 hours after addition of the test compound.
  • the cytotoxic activity during addition of the subject compounds (10 ⁇ M) with respect to A549 cells is shown in the graph.
  • the natural type 2-5A indicates a 3 mer, 2′,5′-oligoadenylate with a 5′-monophosphate group having the structure shown below (Imai, J. and Torrence, P. F., J. Org. Chem., 1985, 50(9), 1418-1426).
  • MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
  • MTT/RPMI1640 concentration 5 mg/ml
  • UV absorbance at 540 nm was measured to determine the relative ratio of the number of viable cells of the compound dose group to the number of viable cells of an untreated cell group followed by calculation of the IC 50 concentration that inhibits cell growth by 50%.
  • the table shows the 50% growth inhibitory concentrations of the subject compounds with respect to A549 cells.
  • Example 2 TABLE 50% Growth inhibitory concentrations of the subject compounds with respect to A549 cells Experiment Experiment Experiment 1 2 3 4 Example 2 1.53 0.91 Example 4 0.48 0.61 0.38 Example 5 0.40 0.32 Example 8 2.23 Example 9 1.39 Example 13 2.54 Example 14 2.34 Example 15 0.061 0.073 Example 16 0.09 Example 17 0.35 Example 19 13 Example 22 0.33 Example 23 0.13 0.091 Example 24 0.30 Example 25 0.91 Example 26 0.15 Example 27 0.36 Example 28 0.13 Example 29 0.14 Example 30 0.43 Example 33 2.15 Example 34 6.60
  • the compounds of the present invention have stability and excellent activity (particularly antitumor activity), and are useful as pharmaceutical drugs (particularly antitumor agents).
  • the compounds of the present invention can be administered to a mammal, such as a human, to treat a tumor or a viral disease.

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US20100035976A1 (en) * 2002-11-19 2010-02-11 Sankyo Company, Limited Method of treating a tumor or a viral disease by administering a 2' , 5' -oligoadenylate analog
US20160068561A9 (en) * 2012-06-18 2016-03-10 Daiichi Sankyo Company, Limited Intermediate for Production of Nucleoside Analog and Method for Producing the Same

Families Citing this family (8)

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US20020055479A1 (en) 2000-01-18 2002-05-09 Cowsert Lex M. Antisense modulation of PTP1B expression
US7179796B2 (en) 2000-01-18 2007-02-20 Isis Pharmaceuticals, Inc. Antisense modulation of PTP1B expression
US20050106101A1 (en) * 2003-10-31 2005-05-19 Ajay Purohit Novel chemical agents comprising an adenosine moiety or an adenosine analog moiety and an imaging moiety and methods of their use
SG162743A1 (en) * 2005-05-18 2010-07-29 Mitsui Chemicals Inc Catalyst for olefin polymerization, method for producing olefin polymer, method for producing propylene copolymer, propylene polymer, propylene polymer composition, and use of those
AU2008210411A1 (en) * 2007-01-31 2008-08-07 Alios Biopharma, Inc. 2-5A derivatives and their use as anti-cancer, anti-viral and anti-parasitic agents
US20090181921A1 (en) * 2007-12-21 2009-07-16 Alios Biopharma Inc. 2-5a analogs and their methods of use
WO2009146417A1 (en) * 2008-05-30 2009-12-03 Sigma-Aldrich Co. Compositions and methods for specifically silencing a target nucleic acid
EP2697244B1 (de) 2011-04-13 2017-05-17 Ionis Pharmaceuticals, Inc. Antisense-modulation der ptp18-expression

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989012380A2 (en) 1988-06-09 1989-12-28 Temple University Of The Commonwealth System Of Hi Therapeutic uses of 2',5'-oligoadenylate derivatives
JPH10195098A (ja) 1996-11-18 1998-07-28 Takeshi Imanishi 新規ヌクレオチド類縁体
JPH10304889A (ja) 1997-03-07 1998-11-17 Takeshi Imanishi 新規ビシクロヌクレオシド及びオリゴヌクレオチド類縁体
JP2000297097A (ja) 1999-02-12 2000-10-24 Sankyo Co Ltd 新規ヌクレオシド及びオリゴヌクレオチド類縁体
WO2001022970A1 (en) 1999-09-29 2001-04-05 The Cleveland Clinic Foundation Therapy with 2-5a and interferon
JP2002249497A (ja) 2000-12-21 2002-09-06 Sankyo Co Ltd ２’，５’−オリゴアデニル酸類縁体

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI347948B (en) * 2002-11-19 2011-09-01 Sankyo Co Novel 2',5'-oligoadenylic acid compositions

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989012380A2 (en) 1988-06-09 1989-12-28 Temple University Of The Commonwealth System Of Hi Therapeutic uses of 2',5'-oligoadenylate derivatives
JPH10195098A (ja) 1996-11-18 1998-07-28 Takeshi Imanishi 新規ヌクレオチド類縁体
JPH10304889A (ja) 1997-03-07 1998-11-17 Takeshi Imanishi 新規ビシクロヌクレオシド及びオリゴヌクレオチド類縁体
EP1013661A1 (de) 1997-03-07 2000-06-28 Takeshi Imanishi Bicyclonukleosid- und oligonukleotid-analoga
JP2000297097A (ja) 1999-02-12 2000-10-24 Sankyo Co Ltd 新規ヌクレオシド及びオリゴヌクレオチド類縁体
EP1152009A1 (de) 1999-02-12 2001-11-07 Sankyo Company, Limited Analoga von nukleosiden und oligonukleotiden
JP3420984B2 (ja) 1999-02-12 2003-06-30 三共株式会社 新規ヌクレオシド及びオリゴヌクレオチド類縁体
WO2001022970A1 (en) 1999-09-29 2001-04-05 The Cleveland Clinic Foundation Therapy with 2-5a and interferon
JP2002249497A (ja) 2000-12-21 2002-09-06 Sankyo Co Ltd ２’，５’−オリゴアデニル酸類縁体

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
D. Watling et al., "Analogue inhibitor of 2-5A action: effect on the interferon- mediated inhbition of encephalomyocarditis virus replication," The EMBO. J., vol. 4, No. 2, pp. 431-436, (1985).
English-language International Preliminary Examination Report dated May 12, 2005 of International application PCT/JP2003/014748 filed Nov. 19, 2003 Applicant: Sankyo Company, Ltd.
J. Carpten et al., "Germline mutations in the ribonuclease L gene in families showing linkage with HPC1," Nat. Genetics, vol. 30, No. 2, pp. 181-184, (2002).
Lorraine Rusch, "Caspase-Dependent Apoptosis by 2', 5'-Oligoadenylate Activation of RNase L is Enhanced by IFN-beta," J. Interferon Cytokine Res., 20, pp. 1091-1100, (2000).
Lorraine Rusch, "Caspase-Dependent Apoptosis by 2′, 5′-Oligoadenylate Activation of RNase L is Enhanced by IFN-β," J. Interferon Cytokine Res., 20, pp. 1091-1100, (2000).
Margaret Haugh et al., "Analogues and Analogue Inhibitors of ppp (A2′p) nA Their Stability and Biological Activity,", Eur. J. Biochem., vol. 132, No. 1, pp. 77-84, (1983).
Margaret Haugh et al., "Analogues and Analogue Inhibitors of ppp (A2'p) nA Their Stability and Biological Activity,", Eur. J. Biochem., vol. 132, No. 1, pp. 77-84, (1983).
Mark R. Player et al., "The 2-5A System: Modulation of Viral and Cellular Processes Through Acceleration of RNA Degradation," Pharmacol. Ther., vol. 78, No. 2, pp. 55-113, (1998).
Masamitu Shimazu et al., "Regio- and stereocontrolled synthesis of 2′-5′ linked phosphorothioate oligoadenylates by uranyl ion catalyst in aqueous solution," J. Chem. Soc. Perkin Trans., No. 15, pp. 1778-1785, (2002).
Masamitu Shimazu et al., "Regio- and stereocontrolled synthesis of 2'-5' linked phosphorothioate oligoadenylates by uranyl ion catalyst in aqueous solution," J. Chem. Soc. Perkin Trans., No. 15, pp. 1778-1785, (2002).
Paul F. Torrence et al., "Oligonucleotide Structural Parameters That Influence Binding of 5′-O-Triphosphoadenylyl-(2′-5′)-adenylyl-(2′--5′)-adenosine to the 5′-O- Triphosphoadenylyl-(2′-5′)-adenylyl-(2′--5')-adenosine Dependent Endoribonuclease: Chain Length, Phosphorylation State, and Heterocyclic Base," J. Med. Chem., vol. 27, pp. 726-733, (1984).
Paul F. Torrence et al., "Oligonucleotide Structural Parameters That Influence Binding of 5'-O-Triphosphoadenylyl-(2'-5')-adenylyl-(2'--5')-adenosine to the 5'-O- Triphosphoadenylyl-(2'-5')-adenylyl-(2'--5')-adenosine Dependent Endoribonuclease: Chain Length, Phosphorylation State, and Heterocyclic Base," J. Med. Chem., vol. 27, pp. 726-733, (1984).
Robert W. Sobol et al., "Inhibition of HIV-1 Replication and Activation of RNase L by Phosphorothioate/Phosphodiester 2', 5'-Oligoadenylate Derivatives," J. Biol. Chem., vol. 270, No. 11, pp. 5963-5978, (1995).
Robert W. Sobol et al., "Inhibition of HIV-1 Replication and Activation of RNase L by Phosphorothioate/Phosphodiester 2′, 5′-Oligoadenylate Derivatives," J. Biol. Chem., vol. 270, No. 11, pp. 5963-5978, (1995).
Sharon Naik et al., "RNase L dimerization in a mammalian two-hybrid system in response to 2', 5' -oligoadenylates," Nucl. Acid Res., vol. 26, No. 6, pp. 1522-1527, (1998).
Sharon Naik et al., "RNase L dimerization in a mammalian two-hybrid system in response to 2′, 5′ -oligoadenylates," Nucl. Acid Res., vol. 26, No. 6, pp. 1522-1527, (1998).
Simon S. Jones et al., "Chemical Synthesis of 5' -O-Triphosphoryladenylyl-(2'-5')-adenylyl-(2'-5')-adenosine (2-5A)," J. Ame. Chem. Soc., vol. 24, pp. 7399-7401, (1979).
Simon S. Jones et al., "Chemical Synthesis of 5′ -O-Triphosphoryladenylyl-(2′-5′)-adenylyl-(2′-5′)-adenosine (2-5A)," J. Ame. Chem. Soc., vol. 24, pp. 7399-7401, (1979).
Steven A. Adah et al., "Chemistry and Biochemistry of 2', 5'-Oligoadenylate-Based Antisense Strategy," Curr. Med. Chem., 8, pp. 1189-1212, (2001).
Steven A. Adah et al., "Chemistry and Biochemistry of 2′, 5′-Oligoadenylate-Based Antisense Strategy," Curr. Med. Chem., 8, pp. 1189-1212, (2001).
Susan M. Freier et al., "The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes," Nucleic Acids Research, vol. 25, No. 22, pp. 4429-4443, (1997).
Wei Xiao et al., "Synthesis of a 5′-Thiophosphate Analogue of 2-5A, A Phosphatase Resistant Activator of the 2-5A-Dependent Ribonuclease," Bioorg. Med. Chem. Let., vol. 4, No. 21, pp. 2609-2614, (1994).
Wei Xiao et al., "Synthesis of a 5'-Thiophosphate Analogue of 2-5A, A Phosphatase Resistant Activator of the 2-5A-Dependent Ribonuclease," Bioorg. Med. Chem. Let., vol. 4, No. 21, pp. 2609-2614, (1994).
Ying Xiang et al., "Effects of RNase L Mutations Associated with Prostate Cancer on Apoptosis Induced by 2',5'-Oligoadenylates,", Cancer Res., vol. 63, No. 20, pp. 6795-6801, (2003).
Ying Xiang et al., "Effects of RNase L Mutations Associated with Prostate Cancer on Apoptosis Induced by 2′,5′-Oligoadenylates,", Cancer Res., vol. 63, No. 20, pp. 6795-6801, (2003).
Yoshihito Ueno et al., "Synthesis of the antisense oligonucleotides carrying the modified 2-5A molecules at their 5′-termini and their properties," Nucl. Acid Res., Suppl. No. 2, pp. 45-46, (2002).
Yoshihito Ueno et al., "Synthesis of the antisense oligonucleotides carrying the modified 2-5A molecules at their 5'-termini and their properties," Nucl. Acid Res., Suppl. No. 2, pp. 45-46, (2002).

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US20100035976A1 (en) * 2002-11-19 2010-02-11 Sankyo Company, Limited Method of treating a tumor or a viral disease by administering a 2' , 5' -oligoadenylate analog
US7994152B2 (en) * 2002-11-19 2011-08-09 Sankyo Company, Limited Method of treating a cancer by administering A 2′,5′-oligoadenylate analog
US20160068561A9 (en) * 2012-06-18 2016-03-10 Daiichi Sankyo Company, Limited Intermediate for Production of Nucleoside Analog and Method for Producing the Same
US9738681B2 (en) * 2012-06-18 2017-08-22 Daiichi Sankyo Company, Limited Intermediate for production of nucleoside analog and method for producing the same

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